m6A sequence of CAPRIN1 MOV10 TRA2A

N6-methyladenosine (m6A) is a reversible and dynamic RNA modification in eukaryotes. However, how cells establish cell-specific m6A methylomes is still poorly understood. Here, we developed a computational framework to systematically identify cell-specific trans regulators of m6A through integrating gene expressions, binding targets, and binding motifs of large number of RNA binding proteins (RBPs) with a co-methylation network constructed using large-scale m6A methylomes across diverse cell states. We applied the framework and successfully identified 33 high-confidence m6A regulators that modulated the variable m6A sites away from stop codons in a cell-specific manner. To validate them, we knocked down 3 regulators respectively and found 2 of them (TRA2A and CAPRIN1) selectively promoted the methylations of the m6A sites co-localized with their binding targets on RNAs through physical interactions with the m6A writers. Knockdown of TRA2A increased the stabilities of the RNAs with TRA2A bound near the m6A sites and decreased the viability of cells. The successful identification of m6A regulators demonstrates a powerful and widely applicable strategy to elucidate the cell-specific m6A regulators. Additionally, our discovery of pervasive trans-acting regulating of m6A provides novel insights into the mechanisms by which spatial and temporal dynamics of m6A methylomes are established.

N6-甲基腺嘌呤（N6-methyladenosine，m6A）是真核生物中一种可逆且动态的RNA修饰。然而，细胞如何构建细胞特异性的m6A甲基化组，目前仍不甚明晰。本研究开发了一种计算框架，通过整合大量RNA结合蛋白（RNA binding proteins，RBPs）的基因表达、结合靶点与结合基序，并结合基于跨多种细胞状态的大规模m6A甲基化组构建的共甲基化网络，系统性鉴定细胞特异性的m6A反式调控因子。我们将该框架付诸应用，成功鉴定出33个高可信度的m6A调控因子，这些因子可通过细胞特异性方式调控终止密码子远端的可变m6A位点。为验证这些调控因子，我们分别敲低了3个调控因子，结果发现其中2个（TRA2A与CAPRIN1）可通过与m6A写入酶发生物理相互作用，选择性促进与其在RNA上的结合靶点共定位的m6A位点的甲基化。敲低TRA2A会增强其结合位点邻近m6A位点的RNA的稳定性，并降低细胞的存活率。本次m6A调控因子的成功鉴定，证明了一种可用于阐明细胞特异性m6A调控因子的高效且普适性策略。此外，我们发现的广泛存在的m6A反式调控作用，为解析m6A甲基化组的时空动态构建机制提供了全新视角。